2.6. pH Measurement and Gluconic Acid Quantification

We inoculated 0.5 mL of bacterial overnight cultures at ~1.5 of OD₆₀₀ into 25 mL of NBRIP with 0.5% of calcium phosphate in 125 mL of flask and incubated at 28 °C with shaking at 200 rpm for 4 days. The pH values were recorded with a pH meter. The bacterial cultures were centrifuged at 14,000 rpm for 10 min. The supernatant was filtered with a 0.2 µm filter column for HPLC injection. Gluconic acid was quantified using an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a quaternary pump, autosampler, DAD detector and degasser.

The chromatographic separation of gluconic acid was achieved at ambient temperature using a 300 × 4.6 mm ID, 5 µm particle size Allure Organic Acids column (Restek, Bellefonte, PA, USA). Isocratic elution was carried out using a mobile phase of 100 mM phosphate buffer at 0.5 mL/min, and the retention time was 6.4 min with a 10 μL injection. A UV/Vis detector at 210 nm wavelength was used for detection. Pure gluconic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as a reference.